Manufacture of trans-[tetrachlorobis(1H-indazole)ruthenate (III)] and compositions thereof

ABSTRACT

The present invention relates to the preparation of compositions comprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)]. Synthesis and formulation preparation is detailed. Impurity profiles are also discussed. Compositions herein are useful for anti-cancer applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional patentapplication Ser. No. 62/501,984, filed May 5, 2017, the entirety ofwhich is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention generally relates to chemical synthesis, and particularlyrelates to a method of making an alkali metal salt oftrans-[tetrachlorobis(1H-indazole)ruthenate (III)].

BACKGROUND OF THE INVENTION

Several methods for the preparation of sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] (also known asKP1339, NKP-1339, IT-139, and Na[Ru^(III)Cl₄(Hind)₂]) exist in theliterature. For example, W. Peti et al, Eur. J. Inorg. Chem. 1999,1551-1555 discloses the following synthesis scheme.

In this method, limited solubility of the tetramethylammoniumchloridesalt results in a requirement for high volumes of solvent. Furthermore,there are toxicity concerns regarding the use of tetramethylammoniumsalts. An additional process is described in U.S. Pat. No. 8,362,266.This process provides a method of making the compoundM-trans-[tetrachlorobis(1H-indazole)ruthenate (III)], wherein M is analkali metal cation, said method comprising the steps of: (1) reacting,in an aqueous solution or a mixture of water and a first organic solventwhich is water soluble, indazoliumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] with an inorganicsalt of said alkali metal cation M, to form the compoundM-trans-[tetrachlorobis(1H-indazole)ruthenate (III)] and an inorganicsalt of indazole; and (2) extracting said indazole from saidM-trans-[tetrachlorobis(1H-indazole)ruthenate (III)] with a secondorganic solvent which is not substantially water soluble. This method issummarized in the scheme below.

The method described above is effective; however, the need for theextraction step and related hold times may limit the effective batchsize. Also, the purity of the compound is directly related to the lengthof time that the compound is in the basic, aqueous environment. Overallyields for this method are in the 20-35%. Therefore, a method that doesnot utilize an extraction process, avoids an aqueous basic environment,is high yielding and produces compound with high purity levels is highlydesirable. Furthermore, a methodology that avoids extraction and largeamounts of organic solvents is also desirable. A methodology primarilyfocused on precipitation followed by filtration would satisfy this need.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Purity of IT-139 drug substance in bulk solution prepared andstored refrigerated (2-8° C.) and at room temperature (18-22° C.).

FIG. 2. HPLC chromatogram for IT-139 stored refrigerated (2-8° C.) for18 hours

FIG. 3. HPLC chromatogram for IT-139 stored at room temperature (18-22°C.) for 18 hours

FIG. 4. HPLC chromatogram using HPLC Method #3 of Formula I-b preparedusing previous synthetic methodology disclosed in U.S. Pat. No.8,362,266.

FIG. 5. HPLC chromatogram using HPLC Method #2 of Formula I-b preparedusing previous synthetic method.

FIG. 6. HPLC chromatogram using HPLC Method #3 of Formula I-b preparedusing the synthetic methodology of the present invention.

FIG. 7. HPLC chromatogram using HPLC Method #2 of Formula I-b preparedusing the synthetic methodology of the present invention.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 1. GeneralDescription

As described herein, the present invention provides methods forpreparing alkali metal salts oftrans-[tetrachlorobis(1H-indazole)ruthenate (III)]. Such compoundsinclude those of Formula I.

wherein M is an alkali metal cation.

The present invention provides synthetic intermediates useful forpreparing such compounds.

The present invention also provides methods for the preparation ofcesium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] as depicted inFormula I-a below.

The present invention also provides methods for the preparation ofsodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] as depicted inFormula I-b below.

2. Definitions

It is understood that the terms sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)], KP1339, NKP-1339,IT-139, and Na[Ru^(III)Cl₄(Hind)₂] all represent the same compound(Formula I-b) and may be used interchangeably.

As used herein, the term amorphous refers to a non-crystalline solidthat lacks long-range order.

Compounds of this invention include those described generally above, andare further illustrated by the embodiments, sub-embodiments, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

The term “aliphatic” or “aliphatic group”, as used herein, denotes ahydrocarbon moiety that may be straight-chain (i.e., unbranched),branched, or cyclic (including fused, bridging, and spiro-fusedpolycyclic) and may be completely saturated or may contain one or moreunits of unsaturation, but which is not aromatic. Unless otherwisespecified, aliphatic groups contain 1-20 carbon atoms. In someembodiments, aliphatic groups contain 1-10 carbon atoms. In otherembodiments, aliphatic groups contain 1-8 carbon atoms. In still otherembodiments, aliphatic groups contain 1-6 carbon atoms, and in yet otherembodiments aliphatic groups contain 1-4 carbon atoms. Aliphatic groupsinclude, but are not limited to, linear or branched, alkyl, alkenyl, andalkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon. This includes any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen, or; a substitutable nitrogen of a heterocyclic ring including═N— as in 3,4-dihydro-2H-pyrrolyl, —NH— as in pyrrolidinyl, or═N(R^(†))— as in N-substituted pyrrolidinyl.

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent, saturated or unsaturated, straightor branched C₁₋₁₂ hydrocarbon chain”, refers to bivalent alkylene,alkenylene, and alkynylene chains that are straight or branched asdefined herein.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains three to seven ring members.The term “aryl” may be used interchangeably with the term “aryl ring”.

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted”, whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Monovalent substituents on a substitutable carbon atom of an “optionallysubstituted” group are independently halogen; —(CH₂)₀₋₄Rº; —(CH₂)₀₋₄ORº;—O—(CH₂)₀₋₄C(O)ORº; —(CH₂)₀₋₄CH(ORº)₂; —(CH₂)₀₋₄SRº; —(CH₂)₀₋₄Ph, whichmay be substituted with Rº; —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may besubstituted with Rº; —CH═CHPh, which may be substituted with Rº; —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(Rº)₂; —(CH₂)₀₋₄N(Rº)C(O)Rº; —N(Rº)C(S)Rº;—(CH₂)₀₋₄N(Rº)C(O)NRº₂; —N(Rº)C(S)NRº₂; —(CH₂)₀₋₄N(Rº)C(O)ORº;—N(Rº)N(Rº)C(O)Rº; —N(Rº)N(Rº)C(O)NRº₂; —N(Rº)N(Rº)C(O)ORº;—(CH₂)₀₋₄C(O)Rº; —C(S)Rº; —(CH₂)₀₋₄C(O)ORº; —(CH₂)₀₋₄C(O)SRº;—(CH₂)₀₋₄C(O)OSiRº₃; —(CH₂)₀₋₄OC(O)Rº; —OC(O)(CH₂)₀₋₄SR—, SC(S)SRº;—(CH₂)₀₋₄SC(O)Rº; —(CH₂)₀₋₄C(O)NRº₂; —C(S)NRº₂; —C(S)SRº; —SC(S)SRO,—(CH₂)₀₋₄OC(O)NRº₂; —C(O)N(ORº)Rº; —C(O)C(O)Rº; —C(O)CH₂C(O)Rº;—C(NORº)Rº; —(CH₂)₀₋₄SSRº; —(CH₂)₀₋₄S(O)₂Rº; —(CH₂)₀₋₄S(O)₂ORº;—(CH₂)₀₋₄OS(O)₂Rº; —S(O)₂NRº₂; —(CH₂)₀₋₄S(O)Rº; —N(Rº)S(O)₂NRº₂;—N(Rº)S(O)₂Rº; —N(ORº)Rº; —C(NH)NRº₂; —P(O)₂Rº; —P(O)Rº₂; —O P(O)Rº₂;—OP(O)(ORº)₂; SiRº₃; —(C₁₋₄ straight or branched alkylene)O—N(Rº)₂; or—(C₁₋₄ straight or branched alkylene)C(O)O—N(Rº)₂, wherein each Rº maybe substituted as defined below and is independently hydrogen, C₁₋₆aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, or, notwithstanding the definitionabove, two independent occurrences of Rº, taken together with theirintervening atom(s), form a 3-12-membered saturated, partiallyunsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, which may besubstituted as defined below.

Monovalent substituents on Rº (or the ring formed by taking twoindependent occurrences of Rº together with their intervening atoms),are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)), —(CH₂)₀₋₂OH,—(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN, —N₃,—(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Such divalent substituents on asaturated carbon atom of Rº include ═O and ═S.

Divalent substituents on a saturated carbon atom of an “optionallysubstituted” group include the following: ═O, ═S, ═NNR*₂, ═NNHC(O)R*,═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Divalent substituents that are bound to vicinalsubstitutable carbons of an “optionally substituted” group include:—O(CR₂)₂₋₃O—, wherein each independent occurrence of R* is selected fromhydrogen, C₁₋₆ aliphatic which may be substituted as defined below, oran unsubstituted 5-6-membered saturated, partially unsaturated, or arylring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. A tetravalent substituent that is bound to vicinalsubstitutable methylene carbons of an “optionally substituted” group isthe dicobalt hexacarbonyl cluster represented by

when depicted with the methylenes which bear it.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†) ₂, —C(S)NR^(†) ₂,—C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein each R^(†) isindependently hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences of Rt,taken together with their intervening atom(s) form an unsubstituted3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Protected hydroxyl groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, theentirety of which is incorporated herein by reference. Examples ofsuitably protected hydroxyl groups further include, but are not limitedto, esters, carbonates, sulfonates allyl ethers, ethers, silyl ethers,alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples ofsuitable esters include formates, acetates, proprionates, pentanoates,crotonates, and benzoates. Specific examples of suitable esters includeformate, benzoyl formate, chloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benylbenzoate, 2,4,6-trimethylbenzoate. Examples of carbonates include9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate.Examples of silyl ethers include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, andother trialkylsilyl ethers. Examples of alkyl ethers include methyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allylether, or derivatives thereof. Alkoxyalkyl ethers include acetals suchas methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl,benzyloxymethyl, beta-(trim ethyl silyl)ethoxymethyl, andtetrahydropyran-2-yl ether. Examples of arylalkyl ethers include benzyl,p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and4-picolyl ethers.

Protected amines are well known in the art and include those describedin detail in Greene (1999). Mono-protected amines further include, butare not limited to, aralkylamines, carbamates, allyl amines, amides, andthe like. Examples of mono-protected amino moieties includet-butyloxycarbonylamino (—NHBOC), ethyloxycarbonylamino,methyloxycarbonylamino, trichloroethyloxycarbonylamino,allyloxycarbonylamino (—NHAlloc), benzyloxocarbonylamino (—NHCBZ),allylamino, benzylamino (—NHBn), fluorenylmethylcarbonyl (—NHFmoc),formamido, acetamido, chloroacetamido, dichloroacetamido,trichloroacetamido, phenylacetamido, trifluoroacetamido, benzamido,t-butyldiphenylsilyl, and the like. Di-protected amines include aminesthat are substituted with two substituents independently selected fromthose described above as mono-protected amines, and further includecyclic imides, such as phthalimide, maleimide, succinimide, and thelike. Di-protected amines also include pyrroles and the like,2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.

Protected aldehydes are well known in the art and include thosedescribed in detail in Greene (1999). Protected aldehydes furtherinclude, but are not limited to, acyclic acetals, cyclic acetals,hydrazones, imines, and the like. Examples of such groups includedimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzyl acetal,bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes, semicarbazones,and derivatives thereof.

Protected carboxylic acids are well known in the art and include thosedescribed in detail in Greene (1999). Protected carboxylic acids furtherinclude, but are not limited to, optionally substituted C₁₋₆ aliphaticesters, optionally substituted aryl esters, silyl esters, activatedesters, amides, hydrazides, and the like. Examples of such ester groupsinclude methyl, ethyl, propyl, isopropyl, butyl, isobutyl, benzyl, andphenyl ester, wherein each group is optionally substituted. Additionalprotected carboxylic acids include oxazolines and ortho esters.

Protected thiols are well known in the art and include those describedin detail in Greene (1999). Protected thiols further include, but arenot limited to, disulfides, thioethers, silyl thioethers, thioesters,thiocarbonates, and thiocarbamates, and the like. Examples of suchgroups include, but are not limited to, alkyl thioethers, benzyl andsubstituted benzyl thioethers, triphenylmethyl thioethers, andtrichloroethoxycarbonyl thioester, to name but a few.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as in neutron scattering experiments, as analyticaltools or probes in biological assays.

The expression “unit dosage form” as used herein refers to a physicallydiscrete unit of inventive formulation appropriate for the subject to betreated. It will be understood, however, that the total daily usage ofthe compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular subject or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; activity of specific activeagent employed; specific composition employed; age, body weight, generalhealth, sex and diet of the subject; time of administration, and rate ofexcretion of the specific active agent employed; duration of thetreatment; drugs and/or additional therapies used in combination orcoincidental with specific compound(s) employed, and like factors wellknown in the medical arts.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, refers to variations of ±20% or insome instances±10%, or in some instances±5%, or in some instances±1%, orin some instances±0.1% from the specified value, as such variations areappropriate to perform the disclosed methods.

3. Description of Exemplary Embodiments

3.1 Drug Substance

In certain embodiments, the present compounds are generally preparedaccording to Scheme I set forth below:

In one aspect, the present invention provides methods for preparingcompounds of Formula I according to the steps depicted in Scheme Iabove. In step S-1, ruthenium (III) chloride is reacted with indazole toform the indazolium salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]. This step (S-1) is well known in the art, see Keppler et. al.Inorganic Chemistry, 26, 1987. At step S-2, the indazolium salt isconverted to the cesium salt oftrans-[tetrachlorobis(1H-indazole)ruthenate (III)], Formula I-a, bytreatment with cesium chloride. One skilled in the art will recognizethis as a salt exchange from the indazolium salt to the cesium salt. Atstep S-3, the cesium salt of Formula I-a is converted to the sodium saltof trans-[tetrachlorobis(1H-indazole)ruthenate (III)], Formula I-b, bytreatment with sodium aluminium sulphate. One skilled in the art willrecognize this as a salt exchange from the cesium salt to the sodiumsalt.

In certain embodiments, each of the aforementioned synthetic steps maybe performed sequentially with isolation of each intermediate performedafter each step. Alternatively, each of steps S-1, S-2, and S-3, asdepicted in Scheme I above, may be performed in a manner whereby noisolation of intermediates is performed.

One of ordinary skill in the art will recognize that the steps S-1, S-2,and S-3 involve the preparation of first the indazolium salt, then thecesium salt, then the sodium salt oftrans-[tetrachlorobis(1H-indazole)ruthenate (III)]. Furthermore, U.S.Pat. No. 8,362,266 describes the preparation of Formula I-b directlyfrom the indazolium salt. One aspect of the present invention includesthe preparation of Formula I-a as an intermediate in the synthesis ofFormula I-b. It was discovered that the cesium salt intermediate ispreferred over existing methods because product purity and overall yieldcan be significantly increased over existing methods. Without wishing tobe bound to any particular theory, we believe the reason for thisincrease in yield and purity is due to the difficulty in isolating theindazolium salt of trans-[tetrachlorobis(1H-indazole)ruthenate (III)].We have found that this material is very difficult to isolate as puresubstance free of solvent, as the filtered material possesses residualwater and hydrochloric acid. One proposed degradation pathway of thematerial is shown in Scheme II below.

Scheme II shows the preparation of compound A(mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], which results from thedisplacement of a chlorine atom by a water molecule. The impurity,Compound A, is also known as the aqua complex in the literature. Theproduction of compound A can be limited by exclusion of water ormaintaining an appreciably high concentration of chloride ions. Forexample, Formula I-b is much more stable in sodium chloride orhydrochloric acid solutions than pure water. One skilled in the art willrecognize that maintaining a concentration of chloride ions reduces thechances of displacement of a chloride on the ruthenium complex by water.Furthermore, it was discovered that the rate of aquation (or preparationof Compound A) is greatly increased in basic solutions.

Because the primary degradation product is an aquation reaction,particularly one that is accelerated in basic aqueous solutions, itwould be preferable to avoid reaction steps that involve dissolvingcompounds of Formula I in water.

One embodiment of the invention provides a method of preparing FormulaI-b by preparing indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)], isolating the material by filtration, drying until the materialis 200%-500% by mass of theoretical yield for use in S-2. In otherembodiments, the invention provides a method of preparing Formula I-b bypreparing indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],isolating the material by filtration, drying until the material is245%-425% by mass of theoretical yield for use in S-2.

Another aspect of the invention is the introduction of step S-2 into thepreparation of Formula I-b. Step S-2 involves the preparation of acesium intermediate, Formula I-a. The cesium intermediate wassurprisingly found to be a critical step of the present inventionbecause it can be isolated by precipitation and filtration, can be driedwithout inducing degradation (as observed with the indazolium salt), andthe dry powder is stable at ambient conditions. As stated above,indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] isisolated by filtration as what can best be described as a mud-likesubstance. The stability of this compound is improved by the presence ofhydrochloric acid (chloride ions). Washing the filtrate with polarsolvents (e.g. methanol) also lead to degradation. Therefore, the bestpractice is to prepare the indazoliumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)], isolate byfiltration, and use directly for S-2 without delay. S-2 consists ofmixing indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] andcesium chloride in a suitable solvent. Suitable solvents can be alcoholwith 1 to 5 carbon atoms, a diol with 2-4 carbon atoms, water, ketoneswith 1 to 6 carbon atoms, cyclic ethers containing 4 to 7 carbon atoms,amides with 1 to 4 carbon atoms, DMSO, sulfolane, esters with 4 to 6carbon atoms, chlorinated hydrocarbons with 1 or 2 carbon atoms, liquidaromatic hydrocarbons, nitriles with 2-6 carbon atoms, or mixture ofthereof.

In one aspect of the present invention, S-2 consists of mixingindazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] and cesiumchloride in ethanol and methyl ethyl ketone to provide Formula I-a. Thecesium salt intermediate was collected by filtering the reaction mixtureand washing with ethanol. In some embodiments, S-2 utilizes 1-10equivalents of cesium chloride in the reaction mixture. In otherembodiments, S-2 utilizes 2-4 equivalents of cesium chloride in thereaction mixture. In the preferred embodiment, the present inventionprovides a method of preparing Formula I-b, wherein 2.8 equivalents ofcesium chloride are used in step S-2. Preferred solvents for S-2 areethanol-containing mixtures, and the most preferred are ethanol-methylethyl ketone (MEK) mixtures, due their ability to form a crystalline MEKsolvate of the cesium salt, which aids the purification. The obtainedMEK solvate in this case is afterwards readily transformed to a morestable hydrate form of cesium salt, by a treatment with aqueous ethanol.

Another aspect of the invention is step S-3 which converts the cesiumsalt intermediate (Formula I-a) into the desired sodium salt, FormulaI-b. Previous methodologies to provide Formula I-b, described herein,include treatment of an aqueous solution oftrans-[tetrachlorobis(1H-indazole)ruthenate (III)] with a sodium saltunder basic conditions. As described above, the aqueous, basicconditions lead to degradation into Compound A. To address this issue,we developed step S-3 which converts Formula I-a into Formula I-b bymixing with sodium aluminium sulfate (NaAl(SO₄)₂). This salt exchangewas performed by mixing sodium aluminium sulfate and Formula I-a inwater. The reaction is performed at high concentration such that thereaction mixture is heterogeneous. The driving force for the reaction isthe differential solubilities of sodium aluminium sulfate and cesiumaluminium sulfate. Sodium aluminium sulfate is soluble in water andprovides a source of sodium ions. Cesium aluminium sulfate is insolublein water, and precipitates out of the reaction mixture. Therefore, thecesium counterion is continually removed from the reaction solution,resulting in the formation of Formula I-b. The insoluble cesiumaluminium sulfate and Formula I-b are isolated by filtration. FormulaI-b is dissolved in a suitable solvent and the cesium aluminium sulfateis removed by filtration. Suitable solvents include low molecular weightalcohols (with 1 to 5 carbon atoms), ketones with 3 to 6 carbon atoms,nitriles with 2 to 5 carbon atoms, esters with 3 to 6 carbon atoms,amides with 1 to 4 carbon atoms, water, diols with 1 to 4 carbon atoms,DMSO, sulfolane, water, or a combination of thereof. The most preferredsolvent for solid extraction is acetonitrile. Formula I-b is thenprecipitated with a suitable anti-solvent and recovered by filtration.Suitable anti-solvents include ethers with 3 to 8 carbon atoms, cyclic,acyclic or aromatic hydrocarbons with 5 to 8 carbon atoms, chlorinatedhydrocarbons with 1 to 4 carbon atoms, benzotrifluoride, chlorobenzene,methyl carbonate. The most preferred antisolvent is methyl tert-butylether (MTBE).

In some embodiments, the present invention provides a method ofpreparing Formula I-b, wherein the concentration of sodium aluminumsulfate in step S-3 is 0.5 M to 1.65 M. In the preferred embodiment, thepresent invention provides a method of preparing Formula I-b, whereinthe concentration of sodium aluminum sulfate in step S-3 is 1.1 M.

In some embodiments, the present invention provides a method ofpreparing Formula I-b, wherein the reaction temperature of step S-3 isfrom −5° C. to 50° C. In the preferred embodiment, the present inventionprovides a method of preparing Formula I-b, wherein the reactiontemperature of step S-3 is from 20° C. to 25° C.

In some embodiments, the present invention provides a method ofpreparing Formula I-b, wherein the reaction time of step S-3 is from 12hours to 168 hours. In the preferred embodiment, the present inventionprovides a method of preparing Formula I-b, wherein the reaction time ofstep S-3 is 30 hours.

Yet another aspect of the invention is a purification step in whichresidual cesium is removed from Formula I-b. This process involvesstirring Formula I-b in the presence of 4 Å molecular sieves withmethanol, followed by precipitation with MTBE. Without wishing to bebound to any particular theory, it is believed that cesium atoms have anaffinity for the 4 Å pores present in the molecular sieves. Furthermore,it was discovered that trace solvent impurities can be removed from thedesired product by stirring and washing with an MTBE solution that issaturated with water. Use of this final purification step affords thehighest purity Formula I-b.

Characterization of the ruthenium containing target compounds requiredmultiple techniques. Nuclear magnetic resonance spectroscopy of theruthenium compounds is difficult due to the 5/2 nuclear spin state, thusalternative characterization methods were employed, including HPLC andx-ray diffraction (crystallography). In order to fully characterize thepurity of IT-139, we purposefully prepared a number of compoundsbelieved to be impurities in the final composition of IT-139, namelyCompounds A, C, and D. The identity of the impurities A, C, and D wasconfirmed by x-ray diffraction. Compound B is an unstable complexbelieved to be an intermediate in the formation of Compound C. Thestructure of the impurity compounds are as follows:

Once the impurity compounds were prepared and identified, theirretention times were analyzed by HPLC, such that the identity andpercentage of impurity could be quickly quantified by HPLC analysis.During this process, we observed that the aqua complex, Compound A,resulted in multiple peaks on the HPLC, and that chromatographic profilewould change as a function of time. It was discovered that the aquacomplex was reacting with the acetonitrile in the mobile phase to forman acetonitrile adduct, Compound B, and that this adduct wassubsequently reacting to form a covalent derivative with acetonitrile,Compound C. (See Inorganic Chemistry, 2008, v47, p 6513-6523). Thisreaction is depicted in Scheme III below.

The relative retention times for each compound are listed in the Tablebelow (Table 1):

TABLE 1 HPLC relative retention times for Formula I-b, Compound A,Compound B, Compound C, and Compound D. Complex Compound Label RRTRu^(III)Cl₃(Hind)₂(H₂O) Compound A 1.10 Ru^(III)Cl₃(Hind)(HN═C(Me)ind)Compound C 1.07 Ru^(III)Cl₃(Hind)₂(CH₃CN) Compound B 1.28Ru^(III)Cl₃(Hind)₃ Compound D 1.59 Na[Ru^(III)Cl₄(Hind)₂] Formula I-b1.0

In some embodiments, the relative retention times (RRT) described inTable 1 can be defined by a range. For instance, the RRT of Compound Acan be 1.09+/−0.02, the RRT of Compound B can be 1.28+/−0.02, the RRT ofCompound C can be 1.06+/−0.03, and the RRT of Compound D can be1.59+/−0.03.

Because the aqua complex (Compound A) will rapidly form compounds B andC in the mobile phase for HPLC analysis, the amount of Compound A in asample submitted for HPLC analysis is determined to be the sum of thepeak areas corresponding to Compounds A, B, and C. One benefit of thesynthetic methodology of present invention over other syntheticmethodologies is the high purity level that can be achieved by thepresent invention. Previous methodologies described above provide afinal product (drug substance) containing 4-8% of Compound A as animpurity. As a comparison, less than 2% of compound A is readilyachievable with the present invention. One embodiment of the presentinvention provides a composition comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] and Compound A,wherein there is no more than 2.0% by weight Compound A in thecomposition. One embodiment of the present invention provides acomposition comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] and Compound A,wherein there is no more than 1.0% by weight Compound A in thecomposition. One embodiment of the present invention provides acomposition comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] and Compound A,wherein there is no more than 1.5% by weight Compound A in thecomposition. One embodiment of the present invention provides acomposition comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] and Compound A,wherein there is no more than 0.5% by weight Compound A in thecomposition. One embodiment of the present invention provides acomposition comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] and Compound A,wherein there is no more than 3.0% by weight Compound A in thecomposition.

Another benefit of the present invention over previous syntheticmethodologies is a reduction in the amounts of impurities produced.Previous synthetic methodologies described above (see, e.g., U.S. Pat.No. 8,362,266) were often analyzed using an HPLC method (e.g. HPLCMethod #3, vide infra) that did not resolve the impurities (Compound A,Compound B, and Compound C) from the drug substance (Formula I-b). FIG.4 illustrates the drug substance prepared using other syntheticmethodologies analyzed using HPLC Method #3, while FIG. 5 illustratesthe same drug substance analyzed using an analytical method (HPLC Method#2, vide infra) which resolves Formula I-b from impurities Compound A,Compound B, and Compound C. Consequently, drug substance synthesizedusing other methodologies reported a purity of about 99.5% (analyzedwith HPLC Method #3), however, the same material analyzed using HPLCMethod #2 demonstrated that the purity was actually approximately about76.4% Formula I-b contaminated with about 7.3% Compound A, about 11.0%Compound B, and about 0.36% Compound C. The present invention provides acomposition comprising Formula I-b in a purity of about 99.9% asanalyzed with the HPLC Method #3. The present invention provides acomposition comprising about 96.3% Formula I-b, about 1.1% Compound A,about 1.7% Compound B, and about 0.2% Compound C. The HPLC data isreproduced in the table below (Table 2).

TABLE 2 HPLC Analysis of Formula I-b, Compound A, Compound B, andCompound C prepared using the present invention and the previoussynthetic method. Formula Impurities Synthesis Analysis I-b CompoundCompound Compound Method Method (area %) A (area %) B (area %) C (area%) Previous HPLC 99.5 n/a¹ n/a¹ n/a¹ methodology Method #3 Previous HPLC76.4 7.3 11.0 0.36 methodology Method #2 Present HPLC 99.9 n/a¹ n/a¹n/a¹ Invention Method #3 Present HPLC 96.3 1.1  1.7  0.2 InventionMethod #2 ¹Impurities not resolved from Formula I-b

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],Ru^(III)Cl₃(Hind)₂(H₂O), Ru^(III)Cl₃(Hind)₂(CH₃CN), andRu^(III)Cl₃(Hind)(HN═C(Me)ind).

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],Ru^(III)Cl₃(Hind)₂(H₂O), Ru^(III)Cl₃(Hind)₂(CH₃CN),Ru^(III)Cl₃(Hind)(HN═C(Me)ind) and cesium.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],Ru^(III)Cl₃(Hind)₂(H₂O), Ru^(III)Cl₃(Hind)₂(CH₃CN), andRu^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium,

-   -   wherein:    -   the sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is        not less than about 95.5 weight percentage of the composition,    -   the Ru^(III)Cl₃(Hind)₂(H₂O) is not more than about 1.0 weight        percentage of the composition,    -   the Ru^(III)Cl₃(Hind)₂(CH₃CN) is not more than about 2.5 weight        percentage of the composition,    -   the Ru^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than about 2.0        weight percentage of the composition,    -   and cesium is not more than about 0.5 weight percentage of the        composition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],cesium, and optionally Ru^(III)Cl₃(Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), and Ru^(III)Cl₃(Hind)(HN═C(Me)ind):

-   -   wherein:    -   the sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is        between about 95.5 and about 99.9 weight percentage of the        composition,    -   the Ru^(III)Cl₃(Hind)₂(H₂O) is between about 0 and about 1.0        weight percentage of the composition,    -   the Ru^(III)Cl₃(Hind)₂(CH₃CN) is between about 0 and about 2.5        weight percentage of the composition,    -   the Ru^(III)Cl₃(Hind)(HN═C(Me)ind) is between about 0 and 2.0        about weight percentage of the composition,    -   and cesium is between about 0 and about 0.5 weight percentage of        the composition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],Ru^(III)Cl₃(Hind)₂(H₂O), Ru^(III)Cl₃(Hind)₂(CH₃CN), andRu^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium,

-   -   wherein:    -   the sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is        between about 95.5 and about 99.9 weight percentage of the        composition,    -   the Ru^(III)Cl₃(Hind)₂(H₂O) is between about 0.001 and about 1.0        weight percentage of the composition,    -   the Ru^(III)Cl₃(Hind)₂(CH₃CN) is between about 0.001 and about        2.5 weight percentage of the composition,    -   the Ru^(III)Cl₃(Hind)(HN═C(Me)ind) is between about 0.001 and        2.0 about weight percentage of the composition,    -   and cesium is between about 0.0001 and about 0.5 weight        percentage of the composition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],Ru^(III)Cl₃(Hind)₂(H₂O), Ru^(III)Cl₃(Hind)₂(CH₃CN), andRu^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium,

-   -   wherein:    -   the sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is        between about 95.5 and about 99.9 weight percentage of the        composition,    -   the Ru^(III)Cl₃(Hind)₂(H₂O) is between about 0.001 and about        0.75 weight percentage of the composition,    -   the Ru^(III)Cl₃(Hind)₂(CH₃CN) is between about 0.001 and about        1.5 weight percentage of the composition,    -   the Ru^(III)Cl₃(Hind)(HN═C(Me)ind) is between about 0.001 and        1.25 about weight percentage of the composition,    -   and cesium is between about 0.0001 and about 0.25 weight        percentage of the composition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],Ru^(III)Cl₃(Hind)₂(H₂O), Ru^(III)Cl₃(Hind)₂(CH₃CN), andRu^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium,

-   -   wherein:    -   the sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is        between about 95.5 and about 99.9 weight percentage of the        composition,    -   the Ru^(III)Cl₃(Hind)₂(H₂O) is between about 0.001 and about 0.5        weight percentage of the composition,    -   the Ru^(III)Cl₃(Hind)₂(CH₃CN) is between about 0.001 and about        0.5 weight percentage of the composition,    -   the Ru^(III)Cl₃(Hind)(HN═C(Me)ind) is between about 0.001 and        0.5 about weight percentage of the composition,    -   and cesium is between about 0.0001 and about 0.01 weight        percentage of the composition.

3.2 Drug Product

Additional embodiments of the present invention provide methods forpreparing drug products containing the sodium salt oftrans-[tetrachlorobis(1H-indazole)ruthenate (III)] (i.e. IT-139).

One aspect of the current invention provides a method for preparing asterile, lyophilized drug product containing sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)]. This formulationwould be suitable for administration to a patient. The formulation iscomprised of sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],a pH buffer, and a cryoprotective agent. The general method forproviding said formulation comprises the steps of preparing aqueousbuffer solution, preparing aqueous cryoprotectant solution, dissolutionof sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] in thebuffer solution, addition of the cryoprotectant solution, sterilefiltration (e.g. aseptic filtration), filling of vials under sterileconditions, and lyophilization under sterile conditions. Suitablebuffers include, but are not limited to: citrate, TRIS, acetate, EDTA,HEPES, tricine, and imidazole. The use of a phosphate buffer is possiblebut is not preferred. A preferred aspect of the present invention is theuse of a citric acid/sodium citrate buffer. Suitable cryoprotectiveagents include, but are not limited to: sugars, monosaccarides,disaccharides, polyalcohols, mannitol, sorbitol, sucrose, trehalose,dextran, and dextrose. A preferred aspect of the present invention isthe use of mannitol as the cyroprotecive agent.

As described above, herein, sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] can degrade in waterto Compound A (Scheme II). One skilled in the art will recognize thatlimiting this degradation reaction would be advantageous to obtainingthe highest purity product. It was found that cooling the sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] solution during theformulation process was found to greatly reduce the amount of Compound Apresent in the lyophilized product. In one aspect of the invention, thesodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)]solution iscooled to 4° C. during the formulation process. In another aspect of theinvention, the sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)]solution is cooled to 2-8° C. during the formulation process. In anotheraspect of the invention, the sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] solution is cooled to2-15° C. during the formulation process.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)], asuitable buffer, and mannitol. In some embodiments, a suitable buffercomprises a citrate buffer. For instance, in some embodiments, a citratebuffer comprises sodium citrate and citric acid.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, and mannitol.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol, andmer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)].

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, and mannitol, wherein the sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] is amorphous.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol, andmer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], wherein the sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] is amorphous.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt, wherein thesodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is amorphous.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is between about 0.01 and about 0.4weight percent of the composition,

and cesium is between about 0.00001 and about 0.01 weight percent of thecomposition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is between about 0.01 and about 0.4weight percent of the composition,

and cesium is between about 0.00001 and about 0.01 weight percent of thecomposition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is between about 0.01 and about 0.2weight percent of the composition,

and cesium is between about 0.00001 and about 0.01 weight percent of thecomposition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is between about 0.01 and about 0.40weight percent of the composition, and cesium is between about 0.00001and about 0.01 weight percent of the composition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

the composition is a lyophilized powder,

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is between about 0.01 and about 0.40weight percent of the composition,

and cesium is between about 0.00001 and about 0.01 weight percent of thecomposition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

the composition is a lyophilized powder,

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is between about 0.01 and about 0.3weight percent of the composition,

and cesium is between about 0.00001 and about 0.1 weight percent of thecomposition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is between about 0.01 and about 0.3weight percent of the composition,

and cesium is between about 0.00001 and about 0.1 weight percent of thecomposition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

the composition is a lyophilized powder,

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 11.5to about 14.0 weight percent of the composition,

citric acid is about 43.9 to about 53.7 weight percent of thecomposition,

sodium citrate is about 25.7 to about 23.1 weight percent of thecomposition,

mannitol is about 11.5 to about 14.0 weight percent of the composition,

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is about 0.01 and about 0.3 weightpercent of the composition,

and cesium is between about 0.00001 and about 0.1 weight percent of thecomposition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

the composition is a lyophilized powder,

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 10.2to about 15.3 weight percent of the composition,

citric acid is about 39.0 to about 58.5 weight percent of thecomposition,

sodium citrate is about 20.5 to about 30.8 weight percent of thecomposition,

mannitol is about 10.2 to about 15.3 weight percent of the composition,

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is about 0.01 and about 0.3 weightpercent of the composition,

and cesium is between about 0.00001 and about 0.1 weight percent of thecomposition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],sodium citrate, citric acid, mannitol,mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)], and a cesium salt;

wherein:

the composition is a lyophilized powder,

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 10.2to about 15.3 weight percent of the composition,

mer,trans-[Ru^(III)Cl₃(Hind)₂(H₂O)] is about 0.01 and about 0.3 weightpercent composition,

and cesium is between about 0.00001 and about 0.1 weight percent of thecomposition.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, and sodium citrate;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 49.86weight percent of the composition,

mannitol is about 49.86 weight percent of the composition,

citric acid is about 0.187 weight percent of the composition,

and sodium citrate is about 0.093 weight percentage of the composition.In some such embodiments, the composition is a lyophilized powder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, and sodium citrate;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 40 toabout 60 weight percent of the composition,

mannitol is about 40 to about 60 weight percent of the composition,

citric acid is about 0.01 to about 0.5 weight percent of thecomposition,

and sodium citrate is about 0.001 to about 0.25 weight percentage of thecomposition. In some such embodiments, the composition is a lyophilizedpowder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, and sodium citrate;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 30 toabout 70 weight percent of the composition,

mannitol is about 30 to about 70 weight percent of the composition,

citric acid is about 0.001 to about 1 weight percent of the composition,

and sodium citrate is about 0.0001 to about 1 weight percentage of thecomposition. In some such embodiments, the composition is a lyophilizedpowder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, and Ru^(III)Cl₃(Hind)₂(H₂O);

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 49.86weight percent of the composition,

mannitol is about 49.86 weight percent of the composition,

citric acid is about 0.187 weight percent of the composition,

sodium citrate is about 0.093 weight percentage of the composition,

and Ru^(III)Cl₃(Hind)₂(H₂O) is not more than 0.5 weight percentage ofthe composition. In some such embodiments, the composition is alyophilized powder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, and Ru^(III)Cl₃(Hind)₂(H₂O);

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 40 toabout 60 weight percent of the composition,

mannitol is about 40 to about 60 weight percent of the composition,

citric acid is about 0.01 to about 0.5 weight percent of thecomposition,

sodium citrate is about 0.001 to about 0.25 weight percentage of thecomposition,

and Ru^(III)Cl₃(Hind)₂(H₂O) is about 0 to about 0.5 weight percentage ofthe composition. In some such embodiments, the composition is alyophilized powder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, Ru^(III)Cl₃(Hind)₂(H₂O), andcesium;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 30 toabout 70 weight percent of the composition,

mannitol is about 30 to about 70 weight percent of the composition,

citric acid is about 0.001 to about 1 weight percent of the composition,

sodium citrate is about 0.0001 to about 1 weight percentage of thecomposition,

Ru^(III)Cl₃(Hind)₂(H₂O) is not more than 0.5 weight percentage of thecomposition,

and cesium is not more than 0.25 weight percentage of the composition.In some such embodiments, the composition is a lyophilized powder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, and cesium;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 49.61weight percent of the composition,

mannitol is about 49.86 weight percent of the composition,

citric acid is about 0.187 weight percent of the composition,

sodium citrate is about 0.093 weight percentage of the composition

and cesium is about 0.25 weight percentage of the composition. In somesuch embodiments, the composition is a lyophilized powder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, and cesium;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 40 toabout 60 weight percent of the composition,

mannitol is about 40 to about 60 weight percent of the composition,

citric acid is about 0.01 to about 0.5 weight percent of thecomposition,

sodium citrate is about 0.001 to about 0.25 weight percentage of thecomposition,

and cesium is about 0.1 to about 0.5 weight percentage of thecomposition. In some such embodiments, the composition is a lyophilizedpowder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, and cesium;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 30 toabout 70 weight percent of the composition,

mannitol is about 30 to about 70 weight percent of the composition,

citric acid is about 0.001 to about 1 weight percent of the composition,

sodium citrate is about 0.0001 to about 1 weight percentage of thecomposition,

and cesium is about 0.01 to about 1 weight percentage of thecomposition. In some such embodiments, the composition is a lyophilizedpowder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, Ru^(III)Cl₃(Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), Ru^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] about 46.61weight percent of the composition,

mannitol is about 49.86 weight percent of the composition,

citric acid is about 0.187 weight percent of the composition,

sodium citrate is about 0.093 weight percentage of the composition,

R^(III)Cl₃(Hind)₂(H₂O) is not more than 0.5 weight percentage of thecomposition,

Ru^(III)Cl₃(Hind)₂(CH₃CN) is not more than 1.25 weight percentage of thecomposition,

R^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than 1.0 weight percentage ofthe composition,

and cesium is not more than 0.25 weight percentage of the composition.In some such embodiments, the composition is a lyophilized powder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, R^(III)Cl₃(Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), Ru^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] about between46.61 weight percent of the composition,

mannitol is about 49.86 weight percent of the composition,

citric acid is about 0.187 weight percent of the composition,

sodium citrate is about 0.093 weight percentage of the composition,

R^(III)Cl₃(Hind)₂(H₂O) is not more than 0.5 weight percentage of thecomposition,

Ru^(III)Cl₃(Hind)₂(CH₃CN) is not more than 1.25 weight percentage of thecomposition,

R^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than 1.0 weight percentage ofthe composition,

and cesium is not more than 0.25 weight percentage of the composition.In some such embodiments, the composition is a lyophilized powder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, R^(III)Cl₃(Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), Ru^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 40 toabout 60 weight percent of the composition,

mannitol is about 40 to about 60 weight percent of the composition,

citric acid is about 0.01 to about 0.5 weight percent of thecomposition,

sodium citrate is about 0.001 to about 0.25 weight percentage of thecomposition,

R^(III)Cl₃(Hind)₂(H₂O) is not more than about 0.5 weight percentage ofthe composition,

Ru^(III)Cl₃(Hind)₂(CH₃CN) is not more than about 1.25 weight percentageof the composition,

R^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than about 1.0 weightpercentage of the composition,

and cesium is not more than 0.25 percentage of the composition. In somesuch embodiments, the composition is a lyophilized powder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, R^(III)Cl₃(Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), Ru^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 30 toabout 70 weight percent of the composition,

mannitol is about 30 to about 70 weight percent of the composition,

citric acid is about 0.001 to about 1 weight percent of the composition,

sodium citrate is about 0.0001 to about 1 weight percentage of thecomposition,

R^(III)Cl₃(Hind)₂(H₂O) is not more than about 0.5 weight percentage ofthe composition,

Ru^(III)Cl₃(Hind)₂(CH₃CN) is not more than about 1.25 weight percentageof the composition,

R^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than about 1.0 weightpercentage of the composition,

and cesium is not more than 0.25 percentage of the composition. In somesuch embodiments, the composition is a lyophilized powder.

One embodiment of the present invention provides a compositioncomprising sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)],mannitol, citric acid, sodium citrate, R^(III)Cl₃(Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), Ru^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium;

wherein:

sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] is about 20 toabout 80 weight percent of the composition,

mannitol is about 20 to about 80 weight percent of the composition,

citric acid is about 0.0001 to about 5 weight percent of thecomposition,

sodium citrate is about 0.00001 to about 5 weight percentage of thecomposition,

R^(III)Cl₃(Hind)₂(H₂O) is not more than about 0.5 weight percentage ofthe composition,

R^(III)Cl₃(Hind)₂(CH₃CN) is not more than about 1.25 weight percentageof the composition,

R^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than about 1.0 weightpercentage of the composition,

and cesium is not more than 0.25 percentage of the composition. In somesuch embodiments, the composition is a lyophilized powder.

3.3 Unit Dosage Forms

In some embodiments, the present invention provides a unit dosage formcomprising a formulation or composition described herein. The expression“unit dosage form” as used herein refers to a physically discrete unitof a provided formulation appropriate for the subject to be treated. Itwill be understood, however, that the total daily usage of providedformulation will be decided by the attending physician within the scopeof sound medical judgment. The specific effective dose level for anyparticular subject or organism will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active agent employed; specific formulationemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activeagent employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

Compositions of the present invention can be provided as a unit dosageform. In some embodiments, a vial comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)], mannitol, citricacid, sodium citrate is a unit dosage form.

In some embodiments, the present invention a vial comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)], mannitol, citricacid, sodium citrate, and cesium is a unit dosage form.

In some embodiments, the present invention a vial comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)], mannitol, citricacid, sodium citrate, Ru^(III)Cl₃ (Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), Ru^(III)Cl₃(Hind)(HN═C(Me)ind), and cesium isa unit dosage form.

Still further encompassed by the invention are pharmaceutical packsand/or kits comprising compositions described herein, or a unit dosageform comprising a provided composition, and a container (e.g., a foil orplastic package, or other suitable container). Optionally instructionsfor use are additionally provided in such kits.

In some embodiments, the present invention can be provided as a unitdosage form. Indeed, a vial comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)], mannitol, citricacid, sodium citrate is a unit dosage form depicted in Table 3

TABLE 3 Pharmaceutical Components Weight Amount/ Component Function %vial sodium trans-[tetra- Active 47.5 100 mg chlorobis(1H-indazole)ruthenate (III)] Mannitol Cryoprotectant 47.5 100 mg Citric Acid Buffer3.37  7.1 mg component Sodium citrate Buffer 1.63  3.4 mg component

In some embodiments, the pharmaceutical components described in Table 3further comprise cesium;

wherein:

cesium is not more than 0.25 weight percentage of the composition.

In some embodiments, the pharmaceutical components described in Table 3further comprise cesium, Ru^(III)Cl₃(Hind)₂(H₂O), Ru^(III)Cl₃(Hind)₂(CH₃CN), and Ru^(III)Cl₃(Hind)(HN═C(Me)ind);

wherein:

cesium is not more than about 0.25 weight percentage of the composition,

Ru^(III)Cl₃(Hind)₂(H₂O) is not more than about 0.5 weight percentage ofthe composition,

Ru^(III)Cl₃(Hind)₂(CH₃CN) is not more than about 1.25 weight percentageof the composition,

and Ru^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than about 1.0 weightpercentage of the composition.

In some embodiments, the pharmaceutical composition is selected fromthose in Table 4:

TABLE 4 Pharmaceutical Component Ranges Weight % Amount/ ComponentFunction Range vial sodium trans-[tetra- Active 42.75-52.25   90-110 mgchlorobis(1H-indazole) ruthenate (III)] Mannitol Cryoprotectant42.75-52.25   90-110 mg Citric Acid Buffer component 3.033-3.7076.39-7.81 mg Sodium citrate Buffer component 1.467-1.793 3.06-3.74 mg

In some embodiments, the pharmaceutical components described in Table 4further comprise cesium;

wherein:

cesium is not more than 0.25 weight percentage of the composition.

In some embodiments, the pharmaceutical components described in Table 4further comprise cesium, Ru^(III)Cl₃ (Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), and Ru^(III)Cl₃(Hind)(HN═C(Me)ind);

wherein:

cesium is not more than about 0.25 weight percentage of the composition,

Ru^(III)Cl₃(Hind)₂(H₂O) is not more than about 0.5 weight percentage ofthe composition,

Ru^(III)Cl₃(Hind)₂(CH₃CN) is not more than about 1.25 weight percentageof the composition,

and Ru^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than about 1.0 weightpercentage of the composition.

In some embodiments, the present invention can be provided as a unitdosage form. Indeed, a vial comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)], mannitol, citricacid, sodium citrate is a unit dosage form depicted in Table 5:

TABLE 5 Pharmaceutical Components Component Function Weight %Amount/vial sodium trans-[tetra- Active 49.86  300 mgchlorobis(1H-indazole) ruthenate (III)] Mannitol Cryoprotectant 49.86 300 mg Citric Acid Buffer component 0.188 1.13 mg Sodium citrate Buffercomponent 0.092 0.55 mg

In some embodiments, the pharmaceutical components described in Table 5further comprise cesium;

wherein:

cesium is not more than 0.25 weight percentage of the composition.

In some embodiments, the pharmaceutical components described in Table 5further comprise cesium, Ru^(III)Cl₃ (Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), and Ru^(III)Cl₃(Hind)(HN═C(Me)ind);

wherein:

cesium is not more than about 0.25 weight percentage of the composition,

Ru^(III)Cl₃(Hind)₂(H₂O) is not more than about 0.5 weight percentage ofthe composition,

Ru^(III)Cl₃(Hind)₂(CH₃CN) is not more than about 1.25 weight percentageof the composition,

and Ru^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than about 1.0 weightpercentage of the composition.

In some embodiments, the pharmaceutical composition is selected fromthose in Table 6:

TABLE 6 Pharmaceutical Components Weight % Amount/ Component FunctionRange vial sodium trans-[tetra- Active  44.87-54.85   270-330 mgchlorobis(1H-indazole) ruthenate (III)] Mannitol Cryoprotectant 44.87-54.85   270-330 mg Citric Acid Buffer  0.169-0.207  1.02-1.24 mgcomponent Sodium citrate Buffer 0.0828-0.1012 0.495-0.605 mg component

In some embodiments, the pharmaceutical components described in Table 6further comprise cesium;

wherein:

cesium is not more than 0.25 weight percentage of the composition.

In some embodiments, the pharmaceutical components described in Table 6further comprise cesium, Ru^(III)Cl₃(Hind)₂(H₂O),Ru^(III)Cl₃(Hind)₂(CH₃CN), and R^(III)Cl₃(Hind)(HN═C(Me)ind);

wherein:

cesium is not more than about 0.25 weight percentage of the composition,

R^(III)Cl₃(Hind)₂(H₂O) is not more than about 0.5 weight percentage ofthe composition,

R^(III)Cl₃(Hind)₂(CH₃CN) is not more than about 1.25 weight percentageof the composition,

and Ru^(III)Cl₃(Hind)(HN═C(Me)ind) is not more than about 1.0 weightpercentage of the composition.

In some embodiments, the pharmaceutical components are as described inany of Tables 3-6, and further comprise cesium. In some embodiments,cesium is present in an amount of about 0.001, 0.002, 0.003, 0.004,0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.015, 0.020, 0.025, 0.030,0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080,0.085, 0.090, 0.095, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45,0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.0weight percentage of the composition.

3.4 Methods of Treatment

In some embodiments, the present invention provides a method fortreating cancer in a subject in need thereof comprising administering tothe subject a provided composition of IT-139 described above and herein.In some such embodiments, the subject is a human patient.

In some embodiments, the present invention provides a method fortreating cancer in a subject in need thereof comprising administering aprovided composition of IT-139 described above and herein in combinationwith a chemotherapeutic agent.

In some embodiments, the present invention provides a method fortreating cancer in a subject in need thereof comprising administering aprovided composition of IT-139 described above and herein in combinationwith an immuno-oncology agent.

According to another embodiment, the present invention relates to amethod of treating a cancer selected from breast, ovary, cervix,prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma,neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoidcarcinoma, large cell carcinoma, small cell carcinoma, lungadenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid,follicular carcinoma, undifferentiated carcinoma, papillary carcinoma,seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma andbiliary passages, kidney carcinoma, myeloid disorders, lymphoiddisorders, Hodgkin's, hairy cells, buccal cavity and pharynx (oral),lip, tongue, mouth, pharynx, small intestine, colon-rectum, largeintestine, rectum, brain and central nervous system, and leukemia,comprising administering IT-139, or a pharmaceutically acceptablecomposition thereof,

According to another embodiment, the present invention relates to amethod of treating a cancer selected from breast, ovary, cervix,prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma,neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoidcarcinoma, large cell carcinoma, small cell carcinoma, lungadenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid,follicular carcinoma, undifferentiated carcinoma, papillary carcinoma,seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma andbiliary passages, kidney carcinoma, myeloid disorders, lymphoiddisorders, Hodgkin's, hairy cells, buccal cavity and pharynx (oral),lip, tongue, mouth, pharynx, small intestine, colon-rectum, largeintestine, rectum, brain and central nervous system, and leukemia,comprising administering IT-139, or a pharmaceutically acceptablecomposition thereof, in combination with a chemotherapeutic agent.

According to another embodiment, the present invention relates to amethod of treating a cancer selected from breast, ovary, cervix,prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma,neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoidcarcinoma, large cell carcinoma, small cell carcinoma, lungadenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid,follicular carcinoma, undifferentiated carcinoma, papillary carcinoma,seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma andbiliary passages, kidney carcinoma, myeloid disorders, lymphoiddisorders, Hodgkin's, hairy cells, buccal cavity and pharynx (oral),lip, tongue, mouth, pharynx, small intestine, colon-rectum, largeintestine, rectum, brain and central nervous system, and leukemia,comprising administering IT-139, or a pharmaceutically acceptablecomposition thereof, in combination with an immuno-oncology agent.

Another embodiment provides a method for treating cancer by reducing theamount of GRP78 in cancer cells following administration of IT-139.

According to another embodiment, the present invention provides a methodfor treating cancer by reducing the amount of GRP78 in cancer cellsfollowing administration of IT-139 in combination with a chemotherapyagent, wherein the administration of IT-139, or a pharmaceuticallyacceptable composition thereof, results in a reduction in the amount ofGRP78 as compared to administration of the chemotherapy agent.

According to another embodiment, the present invention provides a methodfor treating cancer by reducing the amount of GRP78 in cancer cellsfollowing administration of IT-139 in combination with animmune-oncology agent, wherein the administration of IT-139, or apharmaceutically acceptable composition thereof, results in a reductionin the amount of GRP78 as compared to administration of theimmune-oncology agent alone.

The order of administration of therapeutics should be carefullyconsidered. Without wishing to be bound to any particular theory, themechanism of action and down-regulation of GRP78 dictates that anychemotherapeutic agent should be administered first, followed by IT-139for maximum therapeutic benefit. As stated above, treatment with a rangeof chemotherapeutic agents results in an increase ER stress, whichinduces production of GRP78. This process is a cellular survivalmechanism. Administration of IT-139 decreases the level ofstress-induced GRP78, which removes a cellular survival pathway. Theultimate result is increased cancer cell death and increased anti-tumoreffect.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprising the stepsof:

-   -   1) administering to the patient a chemotherapy agent;    -   2) subsequently administering IT-139, or a pharmaceutically        acceptable composition thereof; to the patient; and    -   3) optionally repeating steps 1 and 2.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered 1 day after the chemotherapy agent.In other embodiments, IT-139, or a pharmaceutically acceptablecomposition thereof, is administered to the patient 1 week after thechemotherapy agent. In yet other embodiments, IT-139 is administered toa patient between 1 and seven days after the chemotherapy agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered simultaneously with thechemotherapy agent. In certain embodiments, the IT-139, or apharmaceutically acceptable composition thereof, and the chemotherapyagent are administered within about 20-28 hours of each other, or withinabout 22-26 hours of each other, or within about 24 hours of each other.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered before the chemotherapy agent. Incertain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 8-16 hours beforethe chemotherapy agent, or at least about 10-14 hours before thechemotherapy agent, or at least about 12 hours before the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours beforethe chemotherapy agent, or at least about 22-26 hours before thechemotherapy agent, or at least about 24 hours before the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours beforethe chemotherapy agent, or at least about 46-50 hours before thechemotherapy agent, or at least about 48 hours before the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 64-80 hours beforethe chemotherapy agent, or at least about 70-74 hours before thechemotherapy agent, or at least about 72 hours before the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered before the chemotherapy agent. Incertain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 8-16 hours after thechemotherapy agent, or at least about 10-14 hours after the chemotherapyagent, or at least about 12 hours after the chemotherapy agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours afterthe chemotherapy agent, or at least about 22-26 hours after thechemotherapy agent, or at least about 24 hours after the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours afterthe chemotherapy agent, or at least about 46-50 hours after thechemotherapy agent, or at least about 48 hours after the chemotherapyagent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 64-80 hours afterthe chemotherapy agent, or at least about 70-74 hours after thechemotherapy agent, or at least about 72 hours after the chemotherapyagent.

In certain embodiments, the chemotherapeutic agent is selected from thegroup consisting of gemcitabine, nanoparticle albumin paclitaxel,paclitaxel, docetaxel, cabazitaxel, oxaliplatin, cisplatin, carboplatin,doxorubicin, daunorubicin, sorafenib, everolimus and vemurafenib. Incertain embodiments, the chemotherapeutic agent is gemcitabine.

According to one embodiment of the present invention provides a methodfor treating pancreatic cancer in a patient in need thereof, comprisingthe steps of:

-   -   1) administering a gemcitabine and albumin nanoparticle        paclitaxel;    -   2) subsequently administering IT-139, or a pharmaceutically        acceptable composition thereof; and    -   3) optionally repeating steps 1 and 2.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered simultaneously with gemcitabine. Incertain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, and gemcitabine are administered within about 20-28hours of each other, or within about 22-26 hours of each other, orwithin about 24 hours of each other.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered before gemcitabine. In certainembodiments, the IT-139, or a pharmaceutically acceptable compositionthereof, is administered at least about 8-16 hours before gemcitabine,or at least about 10-14 hours before gemcitabine, or at least about 12hours before gemcitabine.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours beforegemcitabine, or at least about 22-26 hours before gemcitabine, or atleast about 24 hours before gemcitabine.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours beforegemcitabine, or at least about 46-50 hours before gemcitabine, or atleast about 48 hours before gemcitabine.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprisingadministering IT-139, or a pharmaceutically acceptable compositionthereof, in combination with an immuno-oncology agent. In certainembodiments, the immune-oncology agent is administered to the patientprior to the administration of IT-139, or a pharmaceutically acceptablecomposition thereof.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered simultaneously with theimmuno-oncology agent. In certain embodiments, the IT-139, or apharmaceutically acceptable composition thereof, and the immuno-oncologyagent are administered within about 20-28 hours of each other, or withinabout 22-26 hours of each other, or within about 24 hours of each other.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered before the immuno-oncology agent.In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 8-16 hours beforethe immuno-oncology agent, or at least about 10-14 hours before theimmuno-oncology agent, or at least about 12 hours before theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours beforethe immuno-oncology agent, or at least about 22-26 hours before theimmuno-oncology agent, or at least about 24 hours before theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours beforethe immuno-oncology agent, or at least about 46-50 hours before theimmuno-oncology agent, or at least about 48 hours before theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 64-80 hours beforethe immuno-oncology agent, or at least about 70-74 hours before theimmuno-oncology agent, or at least about 72 hours before theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered after the immuno-oncology agent. Incertain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 8-16 hours after theimmuno-oncology agent, or at least about 10-14 hours after theimmuno-oncology agent, or at least about 12 hours after theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 20-28 hours afterthe immuno-oncology agent, or at least about 22-26 hours after theimmuno-oncology agent, or at least about 24 hours after theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 44-52 hours afterthe immuno-oncology agent, or at least about 46-50 hours after theimmuno-oncology agent, or at least about 48 hours after theimmuno-oncology agent.

In certain embodiments, the IT-139, or a pharmaceutically acceptablecomposition thereof, is administered at least about 64-80 hours afterthe immuno-oncology agent, or at least about 70-74 hours after theimmuno-oncology agent, or at least about 72 hours after theimmuno-oncology agent.

In certain embodiments, the immune-oncology agent is selected from thegroup consisting of cytokines, checkpoint inhibitors and antibodiesother than PD-1 antibodies. In certain embodiments, the immune-oncologyagent is selected from the group consisting of interferon, interleukin,PD-L1 antibodies, alemtuzumab, ipilimumab, ofatumumab, atezolizumab andrituximab.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprisingadministering IT-139, or a pharmaceutically acceptable compositionthereof, in combination with a PD-1 antibody. In certain embodiments,the PD-1 antibody is administered prior to the administration of theIT-139, or a pharmaceutically acceptable formulation thereof.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprisingadministering IT-139, or a pharmaceutically acceptable compositionthereof, in combination with a PD-L1 antibody. In certain embodiments,the PD-L1 antibody is administered prior to the administration of theIT-139, or a pharmaceutically acceptable formulation thereof.

According to one embodiment of the present invention provides a methodfor treating cancer in a patient in need thereof, comprisingadministering IT-139, or a pharmaceutically acceptable compositionthereof, in combination with an immune-oncology agent other than a PD-1antibody. In certain embodiments, the immune-oncology agent other than aPD-1 antibody is administered prior to the administration of the IT-139,or a pharmaceutically acceptable formulation thereof.

EXEMPLIFICATION

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It will be understoodthat these examples are for illustrative purposes only and are not to beconstrued as limiting this invention in any manner.

Analytical Methods

The following analytical methods were utilized to characterize thecompounds of the present invention.

HPLC Method 1

Assay and identity of sodium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] was determined by high pressure liquid chromatographywith UV detection at 292 nm. IT-139 drug product was dissolved in waterat concentration of 1 mg/mL, and 10 μL was injected onto an AgilentZorbax SB-C18 (3 μm, 4.6×150 mm) HPLC column. Mobile phase A consistedof 0.1% trifluoroacetic acid in water and mobile phase B consisted of0.1% trifluoroacetic acid in acetonitrile. Separation was achieved bygradient flow at 1.0 mL/minute such that mobile phase A constitutingfrom 90% to 10% from time zero to 12 minutes, with mobile phase Bconstituting from 10% to 90% over 12 minutes. The gradient was thenreversed from 10% mobile phase A and 90% mobile phase B to 90% A and 10%B from 12 minutes to 13 minutes. This continued until the end of the runat 15 minutes. The analyte retention time was 7.7 minutes. Sampletemperature was maintained at 5° C., and column temperature wasmaintained at 25° C.

HPLC Method 2

Two HPLC methods are used because two different impurities co-eluteusing HPLC Method 1. Related substances for IT-139 drug product weredetermined by high pressure liquid chromatography with UV detection at292 nm. IT-139 drug product was dissolved in water at concentration of 1mg/mL, and 10 μL was injected onto an Agilent Zorbax SB-C18 (3 μm,4.6×150 mm) HPLC column. Mobile phase A consisted of 0.1%trifluoroacetic acid in water and mobile phase B consisted of 0.1%trifluoroacetic acid in acetonitrile. Separation was achieved bygradient flow at 1.0 mL/minute such that mobile phase A constituted from90% to 10% from time zero to 15 minutes, with mobile phase B constitutedfrom 10% to 90% over 15 minutes. The gradient was held at 10% A and 90%B from 15 minutes to 19.9 minutes. The gradient was then reversed to 90%A and 10% B from 19.9 minutes to 20 minutes, and was held until the endof the run at 26 minutes. Sample temperature was maintained at 5° C.,and column temperature was maintained at 25° C.

HPLC Method 3

Analysis which does not resolve Formula I-b from Compound A, Compound B,and Compound C uses high pressure liquid chromatography with UVdetection at 297 nm. IT-139 drug substance was dissolved in water at aconcentration of 0.5 mg/mL, and 10 μL was injected onto a PhenomenexLuna, Phenyl-Hexyl (150×3 mm×3 μm) HPLC column. Mobile phase consistedof 25% (volume/volume) methanol in 20 mM ammonium acetate buffer with 4mM acetic acid. Separation was achieved by isocratic flow at 1.0 mL/minfor a total run time of 27 mins with the column temperature maintainedat 25° C.

Elemental Analysis

Galbraith Laboratories (Knoxville, Tenn.) performed all elementalanalysis measurements. Carbon, hydrogen, and nitrogen analysis wasperformed using standard operating procedure ME-14, which requires 1-5mg weighed into a tin capsule followed by combustion at 920-980° C. in aPerkinElmer 2400 Series II CHNS/O Analyzer. Sodium and rutheniumanalysis performed by inductively coupled plasma atomic emissionspectrometry using standard operating procedure ME-70 and ICP-OES Optima5300 instrument. Cesium analysis was performed by inductively coupledplasma atomic emission spectrometry using standard operating procedureME-30.

X-Ray Diffraction

X-ray data was collected on a Bruker D8 Venture Single CrystalDiffractometer with PHOTON 100 CMOS Detector, I μS Copper MX source andOxford Cryostream Plus low temperature device or a Bruker Smart Apex2Single Crystal Diffractometer with Copper radiation with roomtemperature data collection.

Example 1—Purification of Ruthenium Chloride

RuCl₃.xH₂O (100.0 g) was combined with conc. HCl 600 mL and ethanol 99%600 mL. The mixture was distilled under air at normal pressure, toreduce the mixture volume below 400 mL. The resulting concentratedruthenium chloride solution remaining in the distillation flask was thencooled to ambient temperature, filtered through a medium porosity glassBuchner funnel, the Buchner funnel and the flask were rinsed with conc.HCl and the combined filtrates were diluted with additional conc. HCl upto about 500 mL total volume.

Example 2—Preparation of the Indazolium Salt

1H-Indazole 300.0 g (2.54 mol; 6.64 eq.) was combined with water (800mL). Concentrated HCl (4 L) was added, the mixture was stirred untildissolved (20 min). This indazole solution was charged into a 15 Ljacketed stirred glass and Teflon reactor, with a large efficientpaddle-shaped stirrer, internal thermo-probe, air-cooled refluxcondenser topped with a gas outlet tube (for HCl gas release) and 0.5L-sized addition funnel with a stem extended with polyethylene tubing.Additional conc. HCl 4.0 L was combined with the indazole solution inthe reactor, and the mixture was stirred and heated until the internaltemperature reached 90° C., the stirring speed was then turned up, to250 rpm, and the temperature was maintained at 90° C. for at least 30min. The solution of RuCl₃ from Example 1 was then carefully addeddropwise over a period of about 5 hours, from the funnel with stemextended by polyethylene tubing, while maintaining rapid stirring at 250rpm. After complete addition, the addition funnel was rinsed down with asmall amount of concentrated HCl (2×50 mL) and the rinses were alsoadded to the reactor. The combined volume of reaction mixture was 9.5 L;the product precipitated in the form of tan microcrystalline flakes.After the complete addition, the reaction was stirred at 250 rpm at 90°C. for additional 10 hours. The reaction mixture was cooled to 25° C.with stirring, transferred through the bottom drain valve into apolyethylene plastic bucket. The precipitated product was collected byfiltration on 3 L medium porosity glass filter funnel. The reactor andthe stirring paddle was washed down with 2 M HCl, the washings wereadded to the material on the filter funnel. The obtained solids werethoroughly rinsed with additional 2 M HCl, about 2 L, and then partiallydried by suction overnight. This provided 598 g of crude indazoliumsalt, wet with residual 2M HCl, as a brown sticky solid. HPLC analysis:Method 1: 97.7%, Method 2: 98.0%.

Example 3—Preparation of the Cesium Salt

A 10 L wide-mouth flask was charged with wet indazolium salt fromExample 2 and solid powdered CsCl 180.0 g (1.07 mol; 2.8 eq.) was added.Pure non-denatured ethanol 99% (1.8 L) was combined with MEK (2.0 L) wascombined with the indazolium salt and CsCl mixture in a 10 L flask. Themixture was mechanically stirred using a wide Teflon paddle, at 22° C.At first for 5 min at 200 rpm followed by high speed stirring for 2hours at 700 rpm. The resulting orange slurry was collected byfiltration using a medium porosity glass Buchner funnel (3 L), thesolids were washed with 99% ethanol thoroughly and the filter cakematerial was partially dried by suction, for about 1 hour. The obtainedmaterial, containing the cesium salt in the form of an orange-coloredMEK solvate intermixed with leftover CsCl, was transferred into a large4 L beaker. One liter of a mixture of 2:1 (v/v) ethanol with water wasadded to the crude Cs salt solid in a beaker. The slurry was stirredmechanically at 350 rpm for 15 minutes in open beaker: during this timethe bright orange color of MEK solvate slurry turned into a cinnamonred-brown color of hydrate. The solids were collected by filtrationusing the same Buchner funnel used previously to filter the Cs salt. Theobtained solids were washed thoroughly with 99% ethanol, about 1 liter.The material was dried by suction and then in vacuo for 14 hours(overnight). Yield was 226.90 g (0.350 mol) of a cinnamon red-brownsolid, HPLC analysis: no free indazole detected, Method 1: 98.6%, Method2: 99.0% pure. Elemental analysis results found: Cs: 21.6%, Ru: 16.6%,Cl: 21.96%. Theoretical values for dihydrate: Cs: 20.5%, Ru: 15.6%, Cl:21.88%. Theoretical values for monohydrate: Cs: 21.1%, Ru: 16.0%, Cl:22.51%. X-ray diffraction analysis of a single crystal from avacuum-dried sample indicated about 50% occupancy density of the twohydrate water molecules in the crystal structure.

Example 4—Preparation of the Sodium Salt

Solid Al₂(SO₄)₃.18H₂O 1000 g (3.0 mol of Al) was gradually added intostirred D.I. water (2.0 L), followed by solid Na₂SO₄ 213.0 g (3.0 mol ofNa). The mixture was stirred to complete dissolution (about 30 min), thetotal solution volume was adjusted to 2.7 L volume by addition of D.I.water. The resulting 1.1 M solution was filtered before use through afine 0.45 micron SteriCup Durapore membrane, to obtain 2.7 L of 1.1Msolution of NaAl(SO₄)₂. This 1.1M NaAl(SO₄)₂ solution was combined with226.9 g of the Cs salt (0.350 mol) from Example 5, in a 4 L beaker.Solid powdered CsCl 6.0 g was added to the mix, to seed the formation ofCs alum. The mixture was stirred magnetically with a large Teflon-coatedrod stirbar for 30 hours at ambient temperature. During this time, thered-brown slurry of the cesium salt turned into coffee-brown blackslurry of IT-139 intermixed with fine white salt-like crystals of cesiumaluminum salts. The solids were collected by filtration, using a mediumporosity Buchner (3 L), the reaction flask and the solids werethoroughly washed with saturated (=1.5M) aqueous Na₂SO₄, about 1.5 Ltotal (in three portions, 3×0.5 L, until the filtrates were colorless),and the solids were dried by suction on Buchner funnel, followed bydrying in vacuo for at least 1 day. The thoroughly dried solids weretransferred into a 2 L wide mouth Erlenmeyer flask with 700 mLacetonitrile. The mixture was stirred mechanically for 15 min. Theresulting orange slurry was filtered, the insoluble sulfate salts wereremoved from the mixture on a medium porosity Buchner funnel. The saltcake was rinsed with additional acetonitrile 300 mL (3×100 mL, untilcolorless) and then discarded. The combined orange filtrates in a 5 Lround flask were diluted with 4 L of MTBE (added in four 1 L portions,with gentle stirring), the flask was set aside for 30 min to completethe precipitation. The precipitated crude Na salt was collected byfiltration, rinsed thoroughly with MTBE (2×0.5 L) and then dried bysuction and in vacuo. The yield was 190.4 g (100% of theory, calculatedas the dihydrate) of a crude product as fluffy brown solid, retainingMTBE in the form of solvate, HPLC purity 98.4% by Method 1. Elementalanalysis demonstrates that this product contains 0.1-0.8 wt % cesium.The structure of the product was confirmed by x-ray diffraction.

Example 5—Removal of Residual Cesium

190.4 g of material from Example 4 was transferred into a dry 10 Lflask. Equal weight of activated 4A molecular sieves powder (191 g), wasadded. [Aldrich 688363-1KG, sodium aluminosilicate, “SYLOSIV A4”manufactured by Grace Davidson]. Methyl ethyl ketone (4.2 L) was addedto the flask and the mixture was stirred mechanically. Methanol (600 mL)was gradually added into the stirred slurry over a 5 min period. Thestirring (800 rpm) was continued for 30 min, at this time nearly alldark brown lumps of material was dissolved. The resulting orange slurrywas filtered through Whatman fiberglass GF-B filter disc placed on topof a fine-porosity glass Buchner porosity funnels. The spent molecularsieves were rinsed with additional MEK 0.4 L (2×200 mL) and discarded.The combined filtrates were precipitated by gradual addition of MTBE 10L with mechanical stirring. The stirring was turned off and the mixturewas set aside to precipitate for 30 minutes. The precipitated productwas collected by filtration (3 L Buchner funnel), rinsed thoroughly withMTBE 1 L (2×0.5 L) and dried by suction, for about 2 hours, until theBuchner funnel was no longer cold. This provided 184 g of purifiedsodium salt. To remove the solvent traces, the purified material wastreated with wet MTBE. 184 g of the purified sodium salt was combinedwith 3.3 L of wet MTBE (water saturated MTBE), in a 5 L wide mouthErlenmeyer and mechanically stirred (200 rpm) for 40 min. The resultingbrown solids were collected by filtration. The solids were rinsed withwet MTBE, dried by suction and then thoroughly dried in vacuo overnight(15 hours). The yield was 176.11 g of a coffee-brown granular heavysolid, 98.7% pure by HPLC. (Method 1) This corresponds to 85% overallyield from RuCl₃.xH₂O (beginning with Example 1). Elemental analysisdetermined that there was 35 to 750 ppm of cesium remaining.

Example 6—Solution Stability Studies

Compound from Example 5 was prepared at room temperature (20° C.) usingroom temperature solutions, and refrigerated (2-8° C.) using cold (2-8°C.) solutions, for a total volume of 500 mL for each. Citric acidsolution was prepared by dissolving 19.2 grams of citric acid in 1 L ofwater. Sodium citrate solution was prepared by dissolving 29.4 grams ofsodium citrate in 1 L of water. Sodium citrate solution was added to thecitric acid solution until the pH was increased from 2.0 to 3.4.Mannitol solution was prepared by dissolving 13.3 g in 200 mL of water.Solutions of Formula I-b were prepared by adding 16.6 mL of citratebuffer to 400 mL water. 3.33 g sodiumtrans[tetrachlorobis(1H-indazole)ruthenate(III)] was added to thesolution and stirred for 10 minutes. 50 mL of mannitol solution wasadded followed by 33.4 mL of water for a final volume of 500 mL IT-139bulk solution. The room temperature (18-22° C.) sample was stirred usinga magnetic stir plate on the laboratory bench, and the refrigeratedsample was stirred using a magnetic stir plate in a refrigerator (2-8°C.). Aliquots of 100 μL were taken from each sample immediately upondissolution (T=0) and at time points of 0.5, 1, 2, 3, 4, 5, 6, 18, 24,32, and 48 hours. Each sample was added to 1.9 mL methanol in an HPLCvial and mixed by vortex. The purity of sodiumtrans[tetrachlorobis(1H-indazole)ruthenate(III)] in the bulk solutionstored at room temperature (18-22° C.) decreased from 96.8% to 12.1%after 18 hours. The purity of sodiumtrans[tetrachlorobis(1H-indazole)ruthenate(III)] in the bulk solutionstored refrigerated (2-8° C.) decreased from 97.05% to 95.4% after 18hours, and to 89.8% after 48 hours. FIG. 1 demonstrates the percentageof sodium trans[tetrachlorobis(1H-indazole)ruthenate(III)] at roomtemperature (18-22° C.) over 18 hours and refrigerated (2-8° C.) over 48hours. Table 7 shows the percentage of sodiumtrans[tetrachlorobis(1H-indazole)ruthenate(III)] (RT 7.7 min) andimpurities (RRT 0.7, 1.9, and total of unspecified RRT) for each samplebased on HPLC peak area. HPLC chromatograms for IT-139 samples storedrefrigerated or at room temperature at the 18 hour time point are shownin FIG. 2 and FIG. 3, respectively.

TABLE 7 HPLC analysis of sodium trans[tetrachlorobis(1H-indazole)ruthenate(III)] stored at 20° C. and at 4° C. analyzed over time. SampleTime % Peak Area % Peak Area % Peak Area % Peak Area Temp. (h) RT 7.7min RRT 1.09 RRT 1.28 Unspecified 20° C. 0 96.8 0.88 2.26 <0.1 20° C.0.5 96.8 1.08 2.07 <0.1 20° C. 1 96.5 1.14 2.28 <0.1 20° C. 2 96.3 1.222.34 0.13 20° C. 3 95.9 1.35 2.55 0.16 20° C. 4 95.4 1.47 2.89 0.25 20°C. 5 94.9 1.58 3.27 0.26 20° C. 6 94.0 1.88 4 0.13 20° C. 18 12.1 25.260.87 1.82 4° C. 0 97.1 0.96 1.91 <0.1 4° C. 0.5 97.3 0.96 1.7 <0.1 4°C. 1 97.0 1.03 1.9 <0.1 4° C. 2 97.0 1.04 1.86 0.14 4° C. 3 96.8 1.070.94 0.16 4° C. 4 96.5 1.13 2.14 0.26 4° C. 5 96.6 1.11 2.2 <0.1 4° C. 696.8 1.09 2.07 <0.1 4° C. 18 95.4 1.45 3.07 0.11 4° C. 24 94.8 1.34 3.76<0.1 4° C. 32 94.9 1.33 3.65 <0.1 4° C. 48 89.8 2.39 7.56 <0.1

Example 7—Preparation of Compound A

Indazole (400 mg, 3.40 mmol, 1 eq.) was dissolved in 2 mM HCl (1.6 L) at80° C. in a large beaker. The solution was cooled to room temperatureprior to the addition of Na[Ru^(III)Cl₄(Hind)₂] (1.8 g, 3.40 mmol, 1eq.) as an aqueous solution (400 mL H₂O). The resulting brownish-redcolored solution was stirred for 5 min and then left to sit withoutstirring. After 1 day crystals began to form at the bottom of thebeaker. After a total of 3 days a significant amount of crystals formedand were collected by vacuum filtration, washed with H₂O (2×350 mL), anddried overnight under reduced pressure to yield Ru^(III)Cl₃(Hind)₂(H₂O)(930 mg, 59.2%) as dark red crystals. The product was suitable for x-raycrystallography, which was used to confirm the structure.

Example 8—Preparation of Compound C

A 100 mL round-bottom flask fitted with a reflux condenser was chargedwith Ru^(III)Cl₃ (Hind)₂(H₂O) (100 mg, 0.217 mmol) and CH₃CN (6 mL). Theresulting dark red suspension was heated to 50° C. After a few hours thematerial solubilized and was left to stir at 50° C. After 4 days,noticeable precipitation had formed in the reaction flask. The crudereaction mixture was centrifuged to yield a dark brown solid, which waswashed with cold Et₂O (3×, isolated each time via centrifugation). Theresulting light brown powder was >95% pure (HPLC analysis). A smallamount (˜40 mg) of the product was dissolved in a minimal amount ofCH₃CN (˜20 mL), sonicated to dissolve, and then sealed in a vial. After1-2 days, diffraction quality red crystals formed and the structure wasconfirmed by x-ray crystallography.

Example 9—Preparation of Compound D

A 50 mL round-bottom flask fitted with a reflux condenser was chargedwith Hind[Ru^(III)Cl₄(Hind)₂] (198 mg, 0.331 mmol) and THF (10 mL). Theresulting brownish-red suspension was sonicated briefly to break-up alarge chunks of material. The reaction mixture was heated to reflux;after 20 mins the material completely solubilized to yield a dark redsolution. After an additional 20 mins at reflux, the reaction mixturewas cooled to room temperature and aliquots of various volumes (0.1-1.0mL were diluted with varying amount of Et₂O or MTBE (1-15 mL). After 1-2days several crystallization trials had produced dark red crystals. Themost successful attempts involved a dilution factor of 1:2-3 (i.e. 1volume of reaction mixture diluted with 2-3 volumes of either Et₂O orMTBE). The crystals were washed with either cold Et₂O or cold MTBEdepending on the anti-solvent used. This yielded red crystals, whichwere used to confirm the structure via x-ray crystallography.

Example 10—IT-139 Formulation Process

IT-139 was prepared chilled using cold (2-8° C.) solutions for a totalvolume of 1 L drug product. Citrate buffer was prepared by adding sodiumcitrate solution (29.4 g/L in water) to citric acid solution (19.2 g/Lin water) until the pH was increased from 2.0 to 3.4. Working citratebuffer solution was then prepared by adding 33 mL citrate buffer in 767mL water. 6.66 grams of sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] was added to 800 mLof cold (2-8° C.) working citrate buffer solution and stirred using amagnetic stir plate and stir bar for 20 minutes while chilling thesolution at 2-8° C. A working solution of mannitol was prepared bydissolving 13.3 g mannitol in 200 mL water. 100 mL of cold (2-8° C.)mannitol solution was added to the 800 mL of dissolved sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] in working citratebuffer, and stirred for 5 minutes while chilling the solution at 2-8° C.100 mL of cold (2-8° C.) water was added to the drug product solutionfor a final volume of 1 L. The IT-139 drug product bulk solution wasfiltered through a 0.22 μm pass through filter and aseptically filledinto lyophilization vials for a final 30 mL fill in a 50 mL clear glassvial. The vials were partially stoppered and loaded into a lyophilizerwith shelves pre-cooled to −5° C. The lyophilization cycle consisted offreezing at −40° C. for 3 hours, primary drying at −10° C. for 15 hoursat 0.1 mbar, followed by −5° C. for 10 hours at 0.1 mbar, and secondarydrying at 5° C. for 2 hours at 0.05 mbar, followed by 10° C. for 2 hoursat 0.05 mbar, followed by 15° C. for 2 hours at 0.05 mbar, followed by20° C. for 2 hours at 0.05 mbar for a total drying time of 36 hours.Vials were fully stoppered, sealed, and stored at −20° C.

Example 11—IT-139 Formulation Process 2

The IT-139 was prepared chilled using cold (2-8° C.) solutions for atotal volume of 2.8 L drug product. Citrate buffer was prepared byadding sodium citrate solution (29.4 g/L in water) to citric acidsolution (19.2 g/L in water) until the pH was increased from 2.0 to 3.4.Working citrate buffer solution was then prepared by adding 9.3 gcitrate buffer to 1960 g water. 36.3 g of sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] was added to 1969.3grams of cold (2-8° C.) working citrate buffer solution and stirredusing a magnetic stir plate and stir bar for 20 minutes while chillingthe solution at 2-8° C. A working solution of mannitol was prepared bydissolving 35 g mannitol in 525 mL water. 525 mL of cold (2-8° C.)mannitol solution was added to the solution of sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] in working citratebuffer, and stirred for 5 minutes while chilling the solution at 2-8° C.300 mL of cold (2-8° C.) water was added to the drug product solutionfor a final volume of 2.8 L. The IT-139 drug product bulk solution wasfiltered through a 0.22 μm pass through filter and aseptically filledinto lyophilization vials for a final 25.1 gram fill in a 50 mL clearglass vial. The vials were partially stoppered and loaded into alyophilizer with shelves pre-cooled to −5° C. The lyophilization cycleconsisted of freezing at −40° C. for 6 hours, primary drying at −10° C.for 50 hours at 0.2 mbar, and secondary drying at 30° C. for 33 hours at0.2 mbar. Vials were backfilled with nitrogen, fully stoppered, sealed,and stored at 4° C.

Example 12—Batch Analysis of IT-139 Drug Substance

Batch analysis data for drug substance comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)],Ru^(III)Cl₃(Hind)₂(H₂O), Ru^(III)Cl₃(Hind)₂(CH₃CN),R^(III)Cl₃(Hind)(HN═C(Me)ind) and cesium is reproduced in Table 8.

TABLE 8 Batch analysis data for drug substance comprising sodiumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)], Ru^(III)Cl₃(Hind)₂(H₂O), Ru^(III)Cl₃(Hind)₂(CH₃CN), Ru^(III)Cl₃(Hind)(HN =C(Me)ind) and cesium. Acceptance Results: Batch Parameter CriteriaCB186/25 1. Characters Appearance Dark brown powder Dark brown powder 2.Identity IR spectrum Conforms to standard — HPLC retention time Conformsto standard Conforms to standard 3. Tests HPLC purity [% area] NLT 95.597.46 (dried basis) Assay Ruthenium [% wt.] 19.12-21.14 — (dried basis)Assay Chlordie [% wt.] 25.0-30.0 28.5 (dried basis) Water content [%wt.] NMT 7.0 6.64 Assay Cesium [ppm] NMT 5000 40 Assay Aluminum [ppm]NMT 100 <5 4. Impurities Indazole [% wt.] NMT 0.25 not detected Impurity(Compound A) at NMT 1.0 0.63 RRT 1.09 (+/− 0.02) Impurity (Compound B)at NMT 2.5 0.93 RRT 1.28 (+/−0.02) Impurity (Compound C) at NMT 2.0 0.66RRT 1.06 (+/− 0.03) Any unspecified NMT 0.5 0.12 impurity [% area] TotalImpurities [% area] NMT 5.0 2.48 5. Residual Solvents Acetonitrile [ppm]NMT 410 Not detected Methanol [ppm] NMT 3000 Not detected Ethanol [ppm]NMT 5000 Not detected tert-Butyl metyl ether [ppm] NMT 5000 815 Methylethyl ketone [ppm] NMT 5000 1562 6. Heavy Metals Os [ppm] NMT 10 — SiReport results — 7. Microbial bioburden Total aerobic microbial NMT 20 —count [CFU/g] Total combined yeast and NMT 5 — mold count [CFU/g]Bacterial endotoxins [EU/mg] NMT 1.0 —

We claim:
 1. A method for preparing a compound of Formula I-a:

comprising a step of reacting indazoliumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] and cesium chloridein a suitable solvent.
 2. A method for preparing a compound of FormulaI-b:

comprising reacting a compound of Formula I-a:

under conditions suitable to effect a salt exchange.
 3. The method ofclaim 1, further comprising isolating the compound of Formula I-a byprecipitation and filtration, to provide an isolated compound of FormulaI-a.
 4. The method of claim 3, further comprising drying the isolatedcompound of Formula I-a, to provide a dried powdered compound of FormulaI-a.
 5. The method of claim 2, wherein the compound of Formula I-a isprepared by reacting indazoliumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] and cesium chloridein a suitable solvent, to provide a synthesized compound of Formula I-a.6. The method of claim 5, further comprising isolating the synthesizedcompound of Formula I-a by precipitation and filtration, to provide anisolated synthesized compound of Formula I-a.
 7. The method of claim 6,further comprising drying the isolated synthesized compound of FormulaI-a, to provide the compound of Formula I-a for preparing the compoundof Formula I-b.
 8. The method of claim 1, wherein the suitable solventis: an alcohol with 1 to 5 carbon atoms, a diol with 2-4 carbon atoms,water, a ketone with 1 to 6 carbon atoms, a cyclic ether containing 4 to7 carbon atoms, an amide with 1 to 4 carbon atoms, DMSO, sulfolane, anester with 4 to 6 carbon atoms, a chlorinated hydrocarbon with 1 or 2carbon atoms, a liquid aromatic hydrocarbon, a nitrile with 2-6 carbonatoms, or a mixture thereof.
 9. The method of claim 5, wherein thesuitable solvent is: an alcohol with 1 to 5 carbon atoms, a diol with2-4 carbon atoms, water, a ketone with 1 to 6 carbon atoms, a cyclicether containing 4 to 7 carbon atoms, an amide with 1 to 4 carbon atoms,DMSO, sulfolane, an ester with 4 to 6 carbon atoms, a chlorinatedhydrocarbon with 1 or 2 carbon atoms, a liquid aromatic hydrocarbon, anitrile with 2-6 carbon atoms, or a mixture thereof.
 10. The method ofclaim 1, wherein the suitable solvent comprises ethanol and methyl ethylketone in an ethanol-methyl ethyl ketone (MEK) mixture, and the step ofreacting indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)]and cesium chloride forms a crystalline MEK solvate of the compound ofFormula I-a.
 11. The method of claim 10, further comprising treating thecrystalline MEK solvate of the compound of Formula I-a with aqueousethanol to provide a hydrate form of the compound of Formula I-a. 12.The method of claim 10, further comprising isolating the compound ofFormula I-a by precipitation and filtration and washing with ethanol, toprovide an isolated compound of Formula I-a.
 13. The method of claim 1,comprising reacting indazoliumtrans-[tetrachlorobis(1H-indazole)ruthenate (III)] with 1-10 equivalentsof cesium chloride.
 14. The method of claim 1, comprising reactingindazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] with 2-4equivalents of cesium chloride.
 15. The method of claim 1, comprisingreacting indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)]with 2.8 equivalents of cesium chloride.
 16. The method of claim 2,comprising mixing the compound of Formula I-a with sodium aluminiumsulfate (NaAl(SO₄)₂) in water.
 17. The method of claim 16, wherein theconcentration of sodium aluminum sulfate in water is 0.5 M to 1.65 M.18. The method of claim 16, wherein the concentration of sodium aluminumsulfate in water is 1.1 M.
 19. The method of claim 16, furthercomprising isolating insoluble cesium aluminium sulfate and the compoundof Formula I-b by filtration, to provide an isolated compound of FormulaI-b.
 20. The method of claim 19, further comprising dissolving theisolated compound of Formula I-b in a suitable dissolution solvent andremoving cesium aluminium sulfate by filtration, to provide a dissolvedcompound of Formula I-b.
 21. The method of claim 20, wherein thedissolution solvent comprises an alcohol with 1 to 5 carbon atoms, aketone with 3 to 6 carbon atoms, a nitrile with 2 to 5 carbon atoms, anester with 3 to 6 carbon atoms, an amide with 1 to 4 carbon atoms,water, a diol with 1 to 4 carbon atoms, DMSO, sulfolane, or acombination of thereof.
 22. The method of claim 20, wherein thedissolution solvent comprises acetonitrile.
 23. The method of claim 22,further comprising precipitating the dissolved compound of Formula I-bwith an antisolvent, to provide a precipitated compound of Formula I-b.24. The method of claim 23, wherein the antisolvent comprises: an etherwith 3 to 8 carbon atoms; cyclic, acyclic or aromatic hydrocarbons with5 to 8 carbon atoms; chlorinated hydrocarbons with 1 to 4 carbon atoms;benzotrifluoride; chlorobenzene; methyl carbonate; or mixtures thereof.25. The method of claim 23, wherein the antisolvent comprises methyltert-butyl ether (MTBE).
 26. The method of claim 16, wherein the mixingis carried out at from 5° C. to 50° C.
 27. The method of claim 16,wherein the mixing is carried out at from 20° C. to 25° C.
 28. Themethod of claim 27, wherein the mixing is carried out for from 12 hoursto 168 hours.
 29. The method of claim 2, further comprising removingresidual cesium from the compound of Formula I-b, by stirring thecompound of Formula I-b in the presence of 4 Å molecular sieves withmethanol, followed by precipitation with methyl tert-butyl ether (MTBE).30. The method of claim 25, further comprising removing residual cesiumfrom the precipitated compound of Formula I-b, by stirring the compoundof Formula I-b in the presence of 4 Å molecular sieves with methanol,followed by precipitation of the compound of Formula I-b with MTBE. 31.Method of claim 30, further comprising stirring and washing the compoundof Formula I-b with an MTBE solution that is saturated with water.